1. Field of the Invention
This invention relates to nucleotide compounds having at least one 2',5'-riboadenylate unit and a terminal morpholinoadenylate unit, which have potentiated biological activity.
2. Description of the Prior Art
Among the enzymes induced by interferon is 2-5A synthetase which, upon activation by certain double-stranded RNAs, converts ATP into a series of unique 2',5'-linked oligoriboadenylates bearing 5'-terminal triphosphate moieties.
2-5A refers to 2',5'-linked oligoriboadenylates of the general formula ppp5'A2'(p5'A).eta.. Different adenosine nucleotide residues of 2-5A are referred to as N-1, N-2, N-3, etc., where N-1 is the 5'-AMP unit comprising the 5'-terminal residue bearing the triphosphate moiety, N-2 is the adjacent downstream 5'-AMP unit, etc. 2-5A synthetase refers to the enzyme that effects the conversion .eta.ATP.fwdarw.ppp5'A2' (p5'A)n.sub.1 +.eta.-1 pp, (.eta..gtoreq.2) and has been variously referred to as 2-5A synthetase, 2-5A polymerase, 2',5'-nucleotidyl transferase, etc. The 2-5A-activated endoribonuclease has also been called RNase F or RNase L.
This generated 2-5A can activate a latent endoribonuclease, RNase L, which degrades RNA with a preference for cleavage after UpA, UpU, or UpG sequences. For this reason, 2-5A is a potent (IC.sub.50 .about.10.sup.-9 M) inhibitor of protein synthesis in cell-free extracts of eukaryotic cells.
The 2-5A system has been implicated in the mechanism of interferon's antiviral action. Thus, interferon induces the 2-5A synthetase in a potential host cell, and double-stranded RNA, generated by virus replication, triggers the enzyme to produce 2-5A which activates RNase L leading to the possible selective degradation of viral mRNA. Accordingly, the 2-5A system may be the primary pathway for the double-stranded RNA-induced inhibition of translation in extracts of interferon-treated mouse L-cells, and elevated levels of 2-5A, sufficient to effect protein synthesis inhibition, have been detected in mouse L-cells treated with interferon and subsequently infected with encephalomyocarditis virus.
In addition, a cell line, NIH-3T3 (clone 1), devoid of RNase L activity, failed to develop an interferon-induced antiviral state against encephalomyocarditis virus infection.
That the 2-5A system may not be limited to involvement in the mechanism of interferon's antiviral action, but may also be involved in cellular regulation and/or differentiation, has been suggested by the findings of high levels of 2-5A synthetase in diverse cell types such as reticulocytes, lymphocytes, lymphoblastoid cells, estrogen-stimulated and withdrawn chick oviduct cells, or cells from dog or mouse liver. Moreover, the 2-5A synthetase may be induced by treatment with agents other than interferon; for instance, dexamethasone in lymphoblastoid cells, dimethyl sulfoxide or sodium butyrate in Moloney sarcoma virus-transformed murine BALB/c cells (25), and dimethyl sulfoxide in cultures of Friend erythroleukemia cells. In this latter case, elevated synthetase levels were associated with erythroid differentiation and were apparently due to interferon production.
The capacity to employ 2-5A or its congeners directly in intact cells would be of great value since it would provide a means to study the biological role of the 2-5A derivatives as agonists or antagonists. In addition, the proposed role of the 2-5A system in the mechanism of interferon's antiviral action and in cell regulation or differentiation suggests its use as a novel approach to the chemotherapy of virus diseases or cancer. Such applications of 2-5A or its derivatives are limited by two established considerations. First, the 2-5A molecule has a relatively short half-life since it is rapidly degraded by a 2',5'-phosphodiesterase. Second, the 2-5A molecule itself, due to its ionic character, cannot penetrate the eukaryotic cell and is devoid of biological activity toward the untreated intact cell.
It was reported by J. C. Khym in Biochemistry 2:344-350 (1963) that he had modified a single nucleoside ribose ring to a morpholine, and a corrected molecular structure of this compound was published by Brown, et al., in J. Chem. Soc. 5072-5074 (1965). This is the most relevant prior art known to the inventors. However, the Khym compound is readily distinguishable from the compounds of this invention because it has (1) only a single ribose converted to a single morpholine, (2) only a methyl substitution at the N of the morpholine, (3) no phosphate moiety, and (4) no biological activity potentiation.
The inventors are also aware of the following U.S. patents, which are listed chronologically and not in relation to relevance.
U.S. Pat. Nos. 3,532,695 and 3,542,776 disclose the modification of periodate oxidized purine ribosides to morpholinoiso-nicotinamides.
U.S. Pat. No. 3,687,808 discloses the replacement of phosphates on a polynucleotide chain by thioanalogs, or linking such analogs to an existing phosphate.
U.S. Pat. No. 3,759,890 discloses imidazoyl derivatives of polysaccharides and polypeptides.
U.S. Pat. No. 3,850,749 discloses oligoribonucleotides including, typically, 2'(3')-O-isovaleryl ApApApA. There is no disclosure of a terminal morpholine, and all compounds have a 3',5'-internucleotide linkage.
U.S. Pat. No. 3,998,822 discloses morpholino-containing pyrido-pyridazines.
U.S. Pat. Nos. 4,000,137 and 4,041,037 disclose various synthetic nucleosides including inosine, adenosine and cytidine useful as antitumorals.
U.S. Pat. No. 4,210,746 discloses pppApApA, but there is no terminal morpholine.
U.S. Pat. No. 4,302,533 discloses (2'-5')pppApApA, but there is no terminal morpholine.